Studies of signal transduction pathways in platelets may improve our comprehension of platelet function and such studies in megakaryocytes can provide information on regulation of their growth and maturation. Dr. Avraham has identified a cytoplasmic tyrosine kinase in human megakaryocytes known as Related Adhesion Focal Tyrosine Kinase (RAFTK). The latter belongs to the focal adhesion kinase (FAK) gene family and is a signal transduction molecule important for normal and neoplastic growth. RAFTK also represents a common link for the action of integrins and mitogenic peptides. Human and murine RAFTK CDNAS have been cloned and characterized. xxxxced amino acid sequences also indicate that RAFTK resembles FAK in that a The megakaryocyte-associated gene will be further characterized with particular reference to a role in signal transduction. The RAFTK will be purified and characterized and its role in signal transduction studied. The functional domains of RAFTK will be mapped and structure-function relationships determined. It will be discerned whether RAFTK expression is required for hematopoietic proliferation and differentiation. In Specific Aim I, RAFTK will be characterized biochemically and immunochemically. The CDNA, encoding domains of RAFTK will be expressed in E. coli. The recombinant proteins expressed will be purified. The CDNA which encodes the full RAFTK will be expressed in a Baculovirus system. The properties of the recombinant protein as a kinase will be studied and monoclonal antibodies to the recombinant RAFTK will be raised. Antibodies recognizing distinct domains in the RAFTK protein will be developed. Reagents which will delineate distinct regulatory domains of RAFTK will be used for further biochemical characterization of the RAFTK protein and its upstream and downstream substrates. In Specific Aim II, participation of RAFTK protein in signal transduction during platelet activation and megakaryocyte proliferation will be delineated. This will involve experiments measuring megakaryocyte-marrow fibroblast adhesion and studies of interactions between megakaryocytes and endothelial cells. Proteins which interact with RAFTK will be identified in these experiments. In Specific Aim III the functional domains of RAFTk will be mapped and structure-function relationships of the protein determined. Sequence components necessary for RAFTK function and localization will be identified. Major tyrosine phosphorylation site(s) will be analyzed. Proline-rich amino acid sequences in RAFTK will be defined with regard to functional importance. In Specific Aim IV, it will be determined whether RAFTK expression is required for hematopoietic proliferation and differentiation. The gene locus of RAFTK will be disrupted in embryonic stem cells by homologous recombination. Mice which are incapable of expressing RAFTK will be generated. The phenotype of mice with defects in hematopoietic development will be examined. Thus the targeting experiments in embryonic stem cells will be designed to test the function of RAFTK in development as well as the requirement for RAFTK in hematopoiesis.